Epidermal growth factor receptor (also referred to as EGFR; ErbB-1; and HER1 in humans) is a cell-surface receptor that is activated when it binds to specific ligands, such as epidermal growth factor (EGF) and transforming growth factor α (TGFα). The cDNA sequence corresponding to normal EGF receptor is disclosed in Ullrich et al. Nature 1984 309, 418-425.
Wong et al., Proc Natl Acad Sci USA 1992, 89, 2965-2969 and PCT Application Serial No. PCT/US90/04489 report the genetic alterations associated with rearrangements or deletions of the gene encoding EGFR in five malignant gliomas including the variant EGFR referred to as the Type III mutant EGF receptor (hereinafter EGFRvIII) which is the translation product of a splice variant of the EGFR gene corresponding to a deletion between nucleotides 275-1075 in the EGF receptor cDNA which corresponds to a deletion of the portion of coding sequence encoding the extracellular domain of the receptor corresponding to exons 2 through 7, such that exon 1 is joined to exon 8. The in-frame splice junction formed by the deletion includes a codon that encodes a glycine residue where the two sequences are joined. This glycine residue coding sequence is not found at the corresponding location in either the normal exon 1 coding sequence or the normal exon 8 coding sequence of the normal EGFR gene. The EGFRvIII deletion results in the fusion of what were ordinarily distant sequences to generate a mutated sequence that encodes a novel peptide sequence at this fusion junction.
EGFRvIII is the most frequent, naturally occurring mutant EGFR in human tumors and is particularly prevalent in the brain tumor called glioblastoma multiforme. EGFRvIII has been reported to be present in 56% of glioblastoma tumors and 16% of non-small cell carcinomas of the lung. Moscatello et al. Cancer Res. 1995, 55, 5536-5539 reports that it has also been found to be present in 78% of breast cancers. EGFRvIII was therefore identified as a potentially ideal tumor target because the sequence was not found in any normal tissue.
The deletion corresponding to exons 2 through 7 in which exon 1 is joined to exon 8 is an in frame alteration that creates a codon for a novel glycine at the junction. The amino terminus of the resulting EGFRvIII protein is characterized by the amino acid sequence LEEKKGNYVVTDH, SEQ ID NO:1 where the L represents the first amino acid of the mature protein, and the G is the result of the exon 1 to 8 fusion.
Vaccines comprising peptides corresponding to the EGFRvIII junction have been used to prevent or induce regression of tumors that overexpress EGFRvIII in animal models. The formulation of the vaccine is using the peptide LEEKKGNYVVTDHC SEQ ID NO:2 (in which the terminal cysteine has been added for conjugation purposes) conjugated to immune stimulatory molecule, KLH. The glycine was thought to be a key feature for the recognition of this peptide as foreign by the immune system because it is novel, although without any direct experimental evidence. This particular peptide:KLH conjugate vaccine has now been used in a Phase II clinical trial for glioblastoma where it was shown median survival of 26 months as compared to 15 months for matched historical controls.
Peptides based upon the protein sequence encoded by the splice junction have been described in U.S. Pat. Nos. 6,224,868, 5,212,290, 5,401,828, 5,710,010, 5,814,317, 5,981,725, 6,127,126 and 6,455,498. Additionally, these patents disclose peptides conjugated to carriers such as keyhole limpet hemocyanin (KLH) and their use as vaccines. Sampson et al. US Publication No. 20090220551 discloses EGFRvIII peptides with different C termini and generally discloses methods of using the peptides as adjuvant therapy in cancer treatment protocols.
Heimberger and Sampson, Expert Opin Biol Ther. 2009 August; 9(8): 1087-1098 disclose results from three different clinical trials using an EGFRvIII-KLH conjugate which comprises the EGFRvIII peptide having SEQ ID NO:2 conjugated to KLH to treat patients with glioblastoma. In one trial, patients first had tumors resected followed by radiation therapy. Thereafter, dendritic cells (DCs) were isolated from patients, pulsed with EGFRvIII-KLH conjugate and reintroduced into patients as autologous DCs. In a second trial, patients had tumors resected followed by radiation therapy and then EGFRvIII-KLH conjugate was administered directly to the patients by injection. In the third trial, patients had tumors resected followed by radiation therapy after which EGFRvIII-KLH conjugate was administered directly to the patients by injection while patients underwent chemotherapy using temozolomide (TMZ). In each clinical trial, improvements in time to progress and overall survival were observed compared to historical time to progress and overall survival statistics.
The current vaccine, while showing a significant prolongation in survival, is not curative. Clearly, patients are desirous of treatments that offer the best possible chance at long term survival. As such, there remains a need to provide improved EGFRvIII vaccines to enhance survival. There remains a need for improved compositions and therapies useful to improve clinical outcomes in patients diagnosed with cancer that expresses EGFRvIII.